Dynamic airway narrowing is a central hallmark of asthma and is caused by contraction of airway smooth muscle. The importance of the small GTPase RhoA in airway contraction has been increasingly recognized. However, the factors that regulate its activation state are not well understood. This proposal develops a research program focused on novel RhoA regulators developed in preliminary data. The central goal is to determine which pathways are most relevant to RhoA activation in asthmatic airway smooth muscle, for identification of novel therapeutic candidates. In Aim 1, I pursue my recent finding that mice deficient for the scaffolding protein IQGAP1 have increased airway responsiveness and RhoA activation in airway smooth muscle. Our preliminary data suggest that IQGAP1 is required for recruitment of the RhoGAP p190A-RhoGAP to RhoA, thus normally facilitating inactivation of RhoA. I propose to test this model with biochemical studies aimed at disrupting interaction between IQGAP1 and p190A-RhoGAP and with mouse models of airway contraction involving both IQGAP1 and p190A-RhoGAP. In Aim 2, I will determine the functional relevance of ArhGAP18, a RhoGAP that I have found in preliminary data to be highly expressed in airway smooth muscle. Furthermore, ArhGAP18 expression is decreased in airway biopsies from asthma patients compared with healthy controls. shRNA knockdown in airway smooth muscle cells and airway models with ArhGAP18 knockout mice will be used to query the relevance of this protein to the regulation of airway contractility. In Aim 3, I focus on factors that intensify RhoA activation, known as guanine nucleotide exchange factors, because they could be targeted for therapeutic inhibition. In order to increase disease relevance, we will interrogate airway smooth muscle gene expression in patient airway biopsies and also perform functional testing of the effects of individual candidates on RhoA activation. These three aims are not co-dependent and will advance knowledge about a relatively unexplored pathway, RhoA activation, in airway smooth muscle pathobiology. The studies proposed are mechanistic and employ novel approaches that could have a significant impact on the development of novel therapies for asthma.